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Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells [1]

['Hélène Gaillard', 'Centro Andaluz De Biología Molecular Y Medicina Regenerativa Cabimer', 'Consejo Superior De Investigaciones Científicas Universidad De Sevilla Universidad Pablo De Olavide', 'Seville', 'Departamento De Genética', 'Facultad De Biología', 'Universidad De Sevilla', 'Toni Ciudad', 'Departamento De Ciencias Biomédicas', 'Facultad De Ciencias']

Date: 2024-09

Abstract The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.

Author summary The genome of living organisms is constantly challenged by intrinsic and extrinsic DNA damaging agents. The resulting DNA lesions must be readily repaired to maintain genome integrity. This is particularly important for bulky DNA lesions, such as those produced by UV light, as they will block the progress of elongating RNA polymerases on transcribed genes. These DNA lesions are repaired by a specific pathway called transcription-coupled nucleotide excision repair (TC-NER), the dysfunction of which is associated with severe human diseases. In this work, we used budding yeast as a eukaryotic model organism to perform a genetic screen for new TC-NER factors. We discovered that the HTZ1 gene, encoding the histone variant H2A.Z, is required for efficient DNA repair by TC-NER. Our molecular and genetic analyses showed that in the absence of H2A.Z, RNA polymerases persist on damaged DNA, causing interference with DNA replication and genome instability. Our findings about the contribution of histone variant H2A.Z to the repair of UV damage further highlight the importance of appropriate chromatin environment for the maintenance of genome integrity.

Citation: Gaillard H, Ciudad T, Aguilera A, Wellinger RE (2024) Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells. PLoS Genet 20(9): e1011300. https://doi.org/10.1371/journal.pgen.1011300 Editor: Dmitry A. Gordenin, National Institute of Environmental Health Sciences, UNITED STATES OF AMERICA Received: May 11, 2024; Accepted: August 26, 2024; Published: September 10, 2024 Copyright: © 2024 Gaillard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: Research was funded by the Spanish Ministry of Science, Innovation and Universities (PID2022-140466NB-I00 to HG and RW), the Junta de Andalucía (P20_01220 to RW), the University of Seville (PP2018-10767 and PP2019-13299 to HG), the Spanish Ministry of Science and Innovation (BFU2016-75058-P to AA), the Spanish Ministry of Economy and Competitiveness (BFU2013-42918-P to AA), and the European Regional Development Fund (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Living cells continuously suffer DNA damage that may lead to mutations and genomic instability if left unrepaired. DNA damage may occur by exogenous agents such as ionizing radiation, ultraviolet (UV) light or chemicals or by endogenous factors derived from the cell metabolism. DNA damage generates structural distortions that interfere with basic cellular functions like transcription and replication. Cells possess a number of pathways to keep DNA lesions, transcription and replication stress under control, many of which are highly conserved throughout evolution. Among the repair pathways, nucleotide excision repair (NER) is a versatile repair pathway capable of removing a large variety of structurally unrelated lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidone 6–4 photoproducts, X-ray induced cyclopurines or adducts induced by chemicals such as 4-nitroquinoline oxide (4-NQO), benzo[a]pyrene, N-acetoxy-2-actylaminofluorene, psoralens, etc. These bulky lesions, which lead to RNA polymerase (RNAP) stalling when located on the template strand, can affect gene expression and may have severe consequences for cell function [1]. Furthermore, trapped RNAPs represent an impediment for the replication machinery and can lead to transcription-replication conflicts (TRCs), a primary source of genome instability [2,3]. Hence, bulky DNA lesions exhibit strong mutagenic and thus tumorigenic potential, as exemplified by skin cancer, which is primarily caused by exposure to natural UV light [4]. Transcription-blocking lesions are mainly repaired by transcription-coupled NER (TC-NER), which differs from global genome NER (GG-NER) in the lesion recognition step, while the core repair reaction is common to both sub-pathways. In TC-NER, stalling of the elongating RNAP at the DNA damage promptly triggers the repair reaction on the transcribed strand (TS) of active genes while in GG-NER, a specialized DNA damage recognition complex–which consists of Rad7, Rad16 and Elc1 in budding yeast–improves the detection of the helix-distorting DNA lesions throughout the genome. In eukaryotes, the human Cockayne’s syndrome protein B (CSB) and its yeast homolog Rad26 are among the first proteins to act at DNA damage-stalled RNAPII and contribute to the recruitment of further repair factors [5,6]. Residual TC-NER activity persists in the absence of Rad26 in yeast and has been shown to depend largely on Rpb9, a nonessential subunit of RNAPII [7]. A handful of factors involved in different aspects of transcription, including elongation, formation of export-competent mRNA ribonucleoprotein complexes and termination are required for TC-NER proficiency [8–10]. Recent works have shown that the Elf1/ELOF1 transcription elongation factor functions in TC-NER both in yeast and human cells, where it promotes the recruitment and assembly of further repair factors [11–13]. Interestingly, these studies also revealed a role for ELOF1 in preventing transcription-replication conflicts upon treatment with genotoxic agents. Despite the advances that have been made in our understanding of TC-NER, the actions taking place at a stalled RNAPII during TC-NER and the crosstalk with other nuclear events remain elusive [1,6]. With the aim at identifying new TC-NER factors to shed light on the molecular mechanisms of this repair pathway, we screened the non-essential deletion strain collection from S. cerevisiae for mutations leading to increased sensitivity to 4-NQO. This genetic screen was performed in strains lacking the RAD7 GG-NER gene as a way to enhance the sensitivity of the assay and enrich in new functions involved in TC-NER. We identified HTZ1, which encodes the yeast H2A.Z histone variant, as a gene required for efficient TC-NER and RNAPII clearance following UV irradiation. Importantly, UV-irradiation of H2A.Z-depleted cells leads to an accumulation of double-strand breaks (DSBs), which constitute a major threat to genome stability.

Discussion Using an unbiased classical genetic screening approach, we have identified histone variant H2A.Z as a new player in TC-NER. Our results support a role for this histone variant both in the regulation of RNAPII occupancy and TC-NER repair efficiency upon UV irradiation. Importantly, defective response to UV damage leads to sustained DSBs and genome instability in htz1Δ cells. H2A.Z is the only histone variant that is conserved from yeast to human and, while it is essential for proper mammalian development, it is not essential in budding yeast except in conditions requiring rapid transcriptional activation [27]. Previous studies have linked H2A.Z to the DNA damage checkpoint [28,29], DSB repair [26,30,31], chromosome segregation [32], DSB-free resolution of stalled replication forks [20] and GG-NER in H2A.Z-bearing nucleosomes at a repressed promoter [18]. While the participation of H2A.Z in DSB repair seems to be conserved in higher eukaryotes [33], the mechanisms underlying the function of this histone variant in the maintenance of genome stability remain largely unresolved. The H2A.Z histone variant had not been linked to TC-NER so far, and to our knowledge only one report relates H2A.Z-containing nucleosomes located at an inactive promoter with increased GG-NER efficiency [18]. Increased repair was observed at the repressed MFA2 promoter and relied on improved Gcn5-mediated histone H3 acetylation and recruitment of the Rad14 NER factor. Notably, this function was not observed at the silent HMRa1 locus were H2A.Z is normally not present, suggesting that pre-existing H2A.Z-containing nucleosomes favor repair at repressed loci. Our CPD repair analysis on the constitutively active RPB2 gene, which is not enriched in H2A.Z nucleosomes [22–24], revealed a weak repair delay on the NTS and a strong repair defect on the TS in htz1Δ cells. These defects likely arise because of a lack of H2A.Z incorporation by the SWR1 complex in response to UV damage, as Htz1 relative enrichment increases upon UV irradiation throughout RPB2 and that swr1Δ, htz1Δ and swr1Δ htz1Δ cells are equally sensitive to UV in a GG-NER deficient background. The SWR1 remodeling complex has been shown to bind nucleosome-free DNA and the adjoining nucleosome core particle, enabling discrimination of gene promoters over gene bodies [34,35]. Stalling of elongating RNAPII at DNA damage sites may lead to nucleosome-free regions that could represent binding sites for SWR1, leading to the incorporation of H2A.Z into surrounding nucleosomes. H2A.Z-enriched chromatin, which enhances DNA accessibility [36], could facilitate repair either by increasing the surface available for interaction with NER proteins or by promoting the action of specific factors that would fuel repair. H2A.Z incorporation may also improve the DNA damage signaling or destabilize RNAPII binding by promoting post-translational modifications on histones or other proteins. Indeed, local changes in chromatin environment triggered by H2A.Z incorporation around a stalled RNAPII likely include Gcn5-mediated H3 acetylation, as reported at the repressed MFA2 promoter [18]. This could account for the observed contribution of H2A.Z to NTS repair, as GG-NER on NTS of active genes has been shown to depend on Rad7-Rad16-mediated Gcn5 recruitment and H3 acetylation [37]. Our ChIP analysis indicates that RNAPIIs remain bound at the 5’-UTR region but not in the RPB2 gene body in UV irradiated htz1Δ cells. This data can reflect either that RNAPIIs accumulate at the 5’-UTR due to defective TC-NER and recovery of transcription elongation, or that UV-dependent degradation of promoter-bound RNAPIIs–a critical pathway to avoid DNA-damage induced transcription stress [38]–does not take place correctly in the absence of histone variant H2A.Z. The latter would imply a direct role for this histone variant in the recruitment of the factors involved in UV-induced RNAPII degradation. Even though further investigation would be required to discriminate between these possibilities, the persistence of chromatin-bound RNAPIIs is predicted to lead to increased TRCs which may challenge genome stability. We found that recombination is greatly increased in a reporter system located in head-on orientation with respect to replication and sustained Rad52-foci are observed in UV-irradiated htz1Δ cells. These results are consistent with the idea that TC-NER defects and persistent stalled RNAPII represent a major barrier to replication fork progression. H2A.Z incorporation by Swr1 was shown to be important for the maintenance of DNA integrity during replication stress [20]. We thus propose a model in which H2A.Z incorporation in the vicinity of RNAPII stalled at DNA lesions promotes efficient TC-NER repair by establishing a repair-favorable chromatin environment and, thus, preventing the accumulation of chromatin-bound RNAPII that could cause conflicts with the replication machinery in UV challenged cells (S4 Fig). Worthy of note, other TC-NER factors were shown to function both in promoting efficient repair on transcribed genes and in avoiding harmful collisions between the replication and transcription machineries. For example, ELOF1 (Elf1 in yeast) not only promotes TC-NER but also protects cells against transcription-mediated replication stress upon DNA damage [12,13]. Another example is provided by the chromatin-reorganizing FACT (facilitates chromatin transcription) complex, which is required for the resolution of TRCs that are mediated by R-loops [39] and for proficient TC-NER in human cells [40]. Thus, TC-NER emerges as a process that may interfere with replication progression. Our finding that histone variant H2A.Z is required for efficient TC-NER and contributes to the prevention of UV-induced DSBs highlights the significance of DNA repair-optimized chromatin environment in maintaining genomic stability.

Material and Methods Yeast strains, plasmids and growth conditions All yeast strains and plasmids used in this study are listed in S2 and S3 Tables, respectively. Deletion mutants were obtained from the yeast deletion collection (Euroscarf) or obtained either by genetic crosses or by standard PCR-based gene replacement. For strains yHG122-1, yHG122-2, yHG122-11, gene replacement was achieved using a rad1Δ::LEU2-containing amplicon obtained from strain W839-6B (R. Rothstein). For strains yHG223-4, yHG224-3, yHG225-3 and yHG226-4, gene replacement was achieved using a rad26Δ::HIS3-containing amplicon obtained from strain MGSC102 [41]. For strain yHG240-3, gene replacement was achieved in yHG231-3C using a rad7Δ::URA3-containing amplicon obtained from strain yHG72-1B. Yeast cells were grown to mid-log phase in rich medium (YPAD; 1% yeast extract, 2% peptone, 2% glucose, 0.004% adenine sulfate) or synthetic defined medium (SD; 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% glucose, 0.2% drop-out mix) at 30°C. 4-Nitroquinoline oxide synthetic genetic array screen We conducted our 4-NQO synthetic genetic array screen in 96-well plates. MATa haploids from the yeast KO collection bearing the G418 resistance marker [14](YSC1053, Open Biosystems) were mated in 100 μl liquid YPD with strain yHG84-2A and grown for 3 days at 30°C without shaking. Diploid cells were selected in 1 ml SD-Trp-Ura medium. After 3 days incubation at 30°C, cells were centrifuged, washed with sterile H 2 O, resuspended in 600 μl sporulation media (1% potassium acetate supplemented with amino acids) and incubated for 6 days at 30° with vigorous shaking. Cells were then centrifuged, washed with sterile H 2 O and digested with zymolyase 20T (100 μg/ml) overnight at 30°C with gentle shaking. Spores were then centrifuged, washed once and resuspended in 500 μl sterile H 2 O. Using an automated robot (Hamilton Microlab Star robotics), spores were spotted on SD-Leu and SD-Leu-Ura plates supplemented with 600 μg/ml G418 to select for haploid cells bearing the KO deletion and the rad7Δ mutation, respectively. Plates were supplemented or not with 0.01 μg/ml 4-NQO. Growth was monitored upon incubation at 30°C for 3 days. The entire procedure was repeated for 820 strains which showed an increased sensitivity to 4-NQO when combined with rad7Δ. The 44 final candidates are described in S1 Table. Drug sensitivity assays Yeast cells grown to mid-log phase in YPAD were adjusted to an initial A 600 of 0.5, serially diluted 1:10, and spotted onto plates without or with the indicated genotoxic agents at the indicated concentrations. For UV irradiation, plates were irradiated in a BS03 UV irradiation chamber (Dr. Gröbel UV-Elektronik GmbH) at the indicated doses and incubated in the dark. Images were taken after 3 to 4 days growth at 30°C. Two or more biological replicates were performed for all conditions. Gene- and strand-specific repair assays CPD repair at the RPB2 gene was analyzed as described [42]. Briefly, cells were grown at 30°C in SD medium, irradiated in SD medium lacking amino acids with 150 J/m2 UV-C light (BS03 UV irradiation chamber; Dr. Gröbel UV-Elektronik GmbH), the medium supplemented with amino acids and cells incubated at 30°C in the dark for recovery. Isolated DNA samples were digested with PvuI and NsiI (Roche) and aliquots mock-treated or treated with T4-endonuclease V (T4endoV, Epicentre). DNA was electrophoresed in 1.3% alkaline agarose gels, blotted to Nylon membranes and hybridized with radioactively labelled strand-specific DNA probes, which were obtained by primer extension. Sequences of the primers are listed in S4 Table. Membranes were analyzed and quantified with a PhosphorImager (Fujifilm FLA5100). The remaining intact restriction fragment after treatment with T4endoV corresponds to the fraction of undamaged DNA. The CPD content was calculated using the Poisson expression, -ln (RF a /RF b ), where RF a and RF b represent the intact restriction fragment signal intensities of the T4endoV- and mock-treated DNA, respectively. Repair curves were calculated as the fraction of CPDs removed versus time. The initial damage was set to 0% repair. Chromatin immunoprecipitation Chromatin immunoprecipitation was performed as described [42]. Briefly, cells were grown at 30°C in SD medium, irradiated in SD medium w/o amino acids with 150 J/m2 UV-C light (BS03 UV irradiation chamber; Dr. Gröbel UV-Elektronik GmbH), the medium supplemented with amino acids and the cells incubated at 30°C in the dark for recovery. Cells were broken in a multibead shocker at 4°C in lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH8, 1% Triton X-100, 0.1% sodium deoxycholate) supplemented with 1x Complete Protease Inhibitor Cocktail (Roche) and 1 mM PMSF. Chromatin was sonicated to an average fragment size of 400–500 bp in a Bioruptor (Diagenode). Samples were centrifuged to eliminate cell debris. An aliquot was processed as Input and the chromatin immunoprecipitated overnight at 4°C with anti-Rpb3 RNAPII subunit (1Y26, Neoclone), anti-HA (ab9110, Abcam) or anti-Myc (9E10, Takara Bio) antibodies coated to Protein A or G Dynabeads (Invitrogen). Mouse IgG (SIGMA) coated Dynabeads were used as control. Real-time quantitative PCR was performed using iTaq universal SYBR Green (Biorad) with a 7500 Real-Time PCR machine (Applied Biosystems). Standard curves for all pairs of primers were performed for each analysis. All PCR reactions were performed in triplicate. Relative Rpb3 enrichment was calculated using the formula 2-ΔΔCt = 2-((Ct INPUT target—Ct IP target)—(Ct INPUT control—Ct IP control)). A non-coding region of chromosome V was used as control. Htz1 relative enrichment was calculated by normalization of the Myc-Htz1 IP signal (2-ΔCt) with the HA-H2B IP signal (2-ΔCt) obtained in parallel from the same chromatin extract. The primer sequences are listed in S4 Table. RT-qPCR analysis Cells were grown to log phase at 30°C in SD medium and total RNA purified with RNAeasy kit (Qiagen) according to manufacturer instructions. 700 ng RNA were treated with ezDNase (Invitrogen) and converted to cDNA using SuperScript III Reverse Transcriptase (Invitrogen). Real-time quantitative PCR was performed using iTaq universal SYBR Green (Biorad) with a 7500 Real-Time PCR machine (Applied Biosystems). SCR1 mRNA was used as internal control for comparative Ct calculation. Standard curves for all pairs of primers were performed for each analysis. All PCR reactions were performed in triplicate. The primer sequences are listed in S4 Table. Recombination assays Recombination frequencies were determined as the average value of the median frequencies obtained from at least three independent fluctuation tests with the indicated recombination systems. Each fluctuation test was performed from six independent colonies according to standard procedures [43]. For UV-induced recombination, plates were irradiated with 20 J/m2 UV-C light (BS03 UV irradiation chamber; Dr. Gröbel UV-Elektronik GmbH) prior to incubation. Detection of Rad52-YFP foci Rad52-YFP foci were visualized in cells transformed with plasmid pWJ1213 with a DM600B microscope (Leica) as previously described [44] with minor modifications. Individual transformants were grown to early-log-phase in SD-Leu, irradiated in SD medium lacking amino acids with 50 J/m2 UV-C light (BS03 UV irradiation chamber; Dr. Gröbel UV-Elektronik GmbH), the medium supplemented with amino acids and cells incubated at 30°C in the dark for recovery. Samples were fixed for 10 minutes in 0.1 M K i PO 4 pH 6.4 containing 2.5% formaldehyde, washed twice in 0.1 M K i PO 4 pH 6.6, and resuspended in 0.1 M K i PO 4 pH7.4. At least 60 S/G2 cells were analyzed in each sample. Average values obtained from at least 3 independent transformants are plotted for each condition. Statistical analyses Statistical analyses were performed using GraphPad Prism 7.0. Statistical significance was determined from at least 3 independent biological replicates using either Student’s t-test or Wilcoxon signed-rank test. Two-tailed unpaired Student’s t-test was used for comparison of the means of two different experimental conditions. Two-tailed Wilcoxon signed-rank test was used for the analyses of time course data. Differences with a P-value lower than 0.05 were considered significant. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. The number of independent experiments (n), specific statistical tests and significance are described in the Figure legends.

Acknowledgments We thank A. Nicolas, B. Guillemette and M. Lisby for strains and plasmids.

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